Abstract DETECTION OF GRAYANOTOXINS IN AZALEA HYBRID. Melanie Luikart, Gina Nguyen. This lab attempted to detect the grayanotoxin within the common Cascade Azalea and the Rozalea hybrid. Grayanotoxin is a strong toxin that is known to be poisonous to pets, livestock, and humans. The methods utilized were Soxhlet extraction, ethanol extraction, liquid-liquid extraction, and thin-layer chromatography (TLC). Though TLC showed that compounds had been successfully extracted from the plant material, determination of the toxin was inconclusive. Using two different solvents, the Rozalea and the positive Grayanotoxin control, Cascade Azalea, showed compounds not present in the negative control, Schefflera. Using the first solvent, ethyl acetate-isopropanol-water, a compound was detected with a retardation factor of 0.773 for the Rozalea and 0.746 for the Cascade Azalea. In contrast, no material was seen in this region from the negative control plant, Schefflera. For the second solvent, toluene-ethyl acetate-water, both the Rozalea and the Cascade Azalea showed evidence of a compound at 0.254, which again was not present in the Schefflera. However, the small amount of available standard Grayanotoxin did not allow detection. From the reference literature using silica gel studies, Grayanotoxin appears on TLC using the first solvent at 0.73 and using the second solvent at 0.18. Keywords: Grayanotoxin, Soxhletapparatus, petroleum ether, lipids, ethanol extraction, liquid-liquid extraction, capillary action, thin-layer chromatography, stationary phase, mobile phase, retardation factor.
Figures: Figure 1: Homemade Soxhlet Apparatus
Figure 2: Ethanol Extraction
Figure 3: Liquid-Liquid Extraction
Figure 4: TLC Strips using ethyl acetate-isopropanol-water Solvent with Measured Retardation Factors (from left to right: schefflera, rozalea, cascade)
Figure 5: Sample TLC Strips using toluene-ethyl acetate-water Solvent with Measured Retardation Factors (from left to right: cascade, rozalea, schefflera)
Summary Graphics:
Table 3: Categorized Retardation Factors of Plant Substances Measured in cm*
Plant
Rf 1
Rf 2
Rf 3
Rf 4
Rf 5
Schefflera
0.842
0.302
Rozalea
0.879
0.773
0.636
0.318
Cascade Azalea
0.891
0.746
0.612
0.513
Rf factors calculated based on 8.50 cm strip, although it is unclear whether this length was enough to completely separate all the compounds in the samples due to visible residue at the end of the run.
Table 4: Multiple Trial Retardation Factors of Plant Substances and Grayanotoxin Control using Toluene-ethyl acetate-water solvent in cm
Trial 1
Trial 2
Trial 3
Trial 4
Averages:
Cascade
Front: 6.65
Front: 7.25
Front: 6.60
Front: 6.90
6.85
.286
.489
.228
.295
.210
.255
.489
Rozalea
Front: 6.90
Front: 7.10
Front: 6.80
Front: 6.60
6.85
.290
.536
.394
.500
.272
.360
.197
.253
.377
.518
Schefflera
Front: 6.70
Front: 6.95
Front: 7.05
Front: 6.90
6.90
Grayanotoxin
Front: 7.15
Front: 6.55
Front: 6.85
6.85
Audacity:
Citations
Constantine, G. H. Jr., Sheth, K., Catalfomo, P. (1967). Grayantoxin I. Occurrence in Additional Ericaceae Species. Journal of Pharmaceutical Sciences, Vol. 56, No. 11.
DETECTION OF GRAYANOTOXINS IN AZALEA HYBRID. Melanie Luikart, Gina Nguyen. This lab attempted to detect the grayanotoxin within the common Cascade Azalea and the Rozalea hybrid. Grayanotoxin is a strong toxin that is known to be poisonous to pets, livestock, and humans. The methods utilized were Soxhlet extraction, ethanol extraction, liquid-liquid extraction, and thin-layer chromatography (TLC). Though TLC showed that compounds had been successfully extracted from the plant material, determination of the toxin was inconclusive. Using two different solvents, the Rozalea and the positive Grayanotoxin control, Cascade Azalea, showed compounds not present in the negative control, Schefflera. Using the first solvent, ethyl acetate-isopropanol-water, a compound was detected with a retardation factor of 0.773 for the Rozalea and 0.746 for the Cascade Azalea. In contrast, no material was seen in this region from the negative control plant, Schefflera. For the second solvent, toluene-ethyl acetate-water, both the Rozalea and the Cascade Azalea showed evidence of a compound at 0.254, which again was not present in the Schefflera. However, the small amount of available standard Grayanotoxin did not allow detection. From the reference literature using silica gel studies, Grayanotoxin appears on TLC using the first solvent at 0.73 and using the second solvent at 0.18. Keywords: Grayanotoxin, Soxhletapparatus, petroleum ether, lipids, ethanol extraction, liquid-liquid extraction, capillary action, thin-layer chromatography, stationary phase, mobile phase, retardation factor.
Figures:
Figure 1: Homemade Soxhlet Apparatus
Figure 2: Ethanol Extraction
Figure 3: Liquid-Liquid Extraction
Figure 4: TLC Strips using ethyl acetate-isopropanol-water Solvent with Measured Retardation Factors (from left to right: schefflera, rozalea, cascade)
Figure 5: Sample TLC Strips using toluene-ethyl acetate-water Solvent with Measured Retardation Factors (from left to right: cascade, rozalea, schefflera)
Summary Graphics:
Table 3: Categorized Retardation Factors of Plant Substances Measured in cm*
Table 4: Multiple Trial Retardation Factors of Plant Substances and Grayanotoxin Control using Toluene-ethyl acetate-water solvent in cm
.489
.489
.536
.500
.360
.377
.518
Citations
Constantine, G. H. Jr., Sheth, K., Catalfomo, P. (1967). Grayantoxin I. Occurrence in Additional
Ericaceae Species. Journal of Pharmaceutical Sciences, Vol. 56, No. 11.
(2003, April). Beginners Guide to Soxhlet Extractions. Anabolicminds Archive. Retrieved from
http://www.erowid.org/archive/rhodium/pdf/soxhlet4dummies.pdf